Integrating Ion Mobility-Resolved Tandem Mass Spectrometry and Microcrystal Electron Diffraction for Structural Elucidation

Abstract

Abstract Determining the three-dimensional structures of small molecules in complex mixtures remains challenging as current approaches typically require physical isolation and quantities of material that are excessive for many components. Here we present a reciprocal structure-determination strategy that integrates ion mobility-resolved high-resolution tandem mass spectrometry (IMS-HRMS-MS/MS) with microcrystal electron diffraction (MicroED/3DED), enabling chemically consistent three-dimensional structure elucidation from sub-microgram quantities of material. Analysis of synthetic reaction mixtures revealed multiple stereoisomeric products with distinct ion-mobility and fragmentation behaviour. Mechanistically grounded modelling of fragmentation pathways and collision cross sections constrained viable three-dimensional molecular geometries, while MicroED independently resolved the structure of a crystallographically accessible isomer. Crucially, the gas-phase-derived geometries served directly as molecular replacement search models for MicroED, including for a second reaction product whose diffraction data could not be solved by direct methods alone, thereby demonstrating direct transfer of structural information between mass spectrometry and electron diffraction. Together, IMS-HRMS-MS/MS and MicroED provide complementary structural projections whose integrated interpretation enables structure elucidation of small molecules directly from complex heterogeneous mixtures using minimal amounts of material.